The PI-3 kinase/Akt pathway is a potential mediator of the cardioprotection provided by preconditioning. The primary purpose of this proposal is the identification of the cardioprotective end-effectors that are activated by the PI-3 kinase/Akt pathway. Heat shock factor-1 (HSF-1) is a transcription factor that initiates the expression of heat shock protein 70 (HSP70), a molecular chaperone that has been associated with the cardioprotection provided by ischemic preconditioning. A role for NFkB in ischemic preconditioning has also been demonstrated. HSF-1 and NFkB activation are negatively regulated by GSK-3 and this regulation is abrogated by the phosphorylation of glycogen synthase kinase (GSK)-3b by Akt. The relative role of GSK-3 in the inhibition of HSF-1 and NFkB will be determined. We will also examine an alternative Akt substrate apoptosis signal regulating kinase 1 (ASK1). We will determine the association of Akt with ASK1 and the subsequent Akt mediated phosphorylation of ASK-1 at Ser83, negatively regulating the activation of p38MAPK and Jun kinase and the initiation of apoptosis. This might involve the interaction of ASK1 with 14-3-3 protein. We will also examine the association of Akt with the small heat shock protein, HSP27, which functions as a scaffolding protein for Akt. We will determine the ability of HSP27, in association with Akt, to facilitate the anti-apoptotic function of Akt. The signaling pathways that initiate PI-3 kinase activation during myocardial ischemia and regulate Akt activity have not been elucidated. Disruption of the dystroglycan complex in muscle cells decreases Akt and GSK-3 phosphorylation, initiating apoptosis. We propose that b-dystroglycan (bDG) assembles a complex of signaling molecules that allows an association with and activation of the p85 subunit of PI-3K, initiating Akt phosphorylation. We propose that PI-3 kinase/Akt activation requires endocytosis of this membrane associated the bDG complex. This is analogous to the recent report that ischemic preconditioning is mediated by an endosomal G-protein coupled receptor activation pathway. These studies will afford a molecular understanding of the mechanisms of irreversible ischemic myocardial injury and the end effectors of preconditioning. This may allow the development of pharmacologic modalities for the therapeutic delay of myocardial ischemia and the preservation of myocardium jeopardized by infarction.